Human Factor VIII, also known as antihaemophilia factor or FVIII:C is a human plasma protein consisting of two polypeptides with light chain molecular weight of 80,000 daltons and a heavy chain molecular weight variable from 90,000 to 220,000. It is considered as one of the key cofactors in the coagulation pathway necessary for the conversion of Factor X into its active form Factor Xa. Factor VIII circulates in plasma as a non-covalent complex with von Willebrand Factor (also known as FVIII:RP). Hemophilia, a bleeding disorder is caused due to abnormal levels of Factor VIII. Factor VIII levels below 20% normal may result in hemophilic condition in humans. A drop in the levels of less than 1% of Factor VIII leads to severe bleeding disorder, with spontaneous joint bleeding being the most common symptom.
The structure and biochemistry of recombinant factor VIII have been described previously.
Traditionally, isolation and purification of Factor VIII has been from a plasma derived source (cryoprecipitate). Purification procedures from plasma-derived sources include those exploring the use of immunoaffinity purification using polyclonal and monoclonal antibodies for the purification of FVIII. However, there may be instances where the Factor VIII effluent contains some residual antibody due to leaching from the support matrix, which may result in antigenicity during ultimate use, i.e when introduced into human or animal system. Purification procedures exploring the use of ion exchange chromatography on e.g. agarose beads have also been used for purification of factor VIII from plasma. These methods, however, often suffer from certain levels of contamination of the resulting FVIII:C.
However, purification of Factor VIII from genetically engineered recombinant source has gained importance in the past decade. Protein recovery and concentration of the final product is of utmost importance in the separation of recombinant proteins. The contaminants in recombinantly produced protein may include secreted proteins in the culture medium, media components, cell lysates, unwanted proteins produced by the cells and the nucleic acids.
When purifying a recombinant protein, the aqueous source materials in which the polypeptides of interest are found are furthermore often seen to be contaminated with one or more viruses. Techniques for inactivating viruses in polypeptide mixtures are known in the art, such as e.g. chemical methods, using solvent/detergent solutions, irradiation methods, or thermal methods, but attempts to combine such techniques with known polypeptide purification processes have produced methods with a multiplicity of steps unsuitable for large-volume production. It is also important to exert caution in that the used viral-inactivating agents do not denature the protein or are difficult to separate from the protein of interest. These agents have, however, been either denaturing or difficult to separate from the polypeptide of interest, and have required a special treatment or separation step. Other conventional methods for treating polypeptide-containing preparations for potential viral contamination, such as heat or irradiation, have resulted in either significant denaturation of the polypeptide of interest and/or insufficient inactivation of viruses. Many of the commercially available recombinant Factor VIII products (Advate®, Helixate®, Kogenate FS®, ReFacto®) are made using immunoaffinity chromatography including a detergent for purification and viral inactivation.
In the purification of therapeutic proteins produced by a recombinant DNA technique, it is well known that considerable problems are encountered when trying to reduce the content of DNA and Host Cell Protein (HCP) to the desired very low level.
Nordfang et al. (Thrombosis and Haemostasis 58(4), 1043-1048 (1987) describes a separation using an antibody resin and a buffer containing 50% ethylene glycol and high salt.
The purification of a recombinant protein expressed in mammalian cell system is typically performed in several steps. The different steps are usually separated into capture, intermediate and polishing. The objective of the capture step is two-fold; a) to obtain the target protein in a stable solution form and b) to reduce the volume (i.e. obtaining a solution concentrated with respect to protein content compared to the solution loaded onto the column (“the loading”).
The latter step (reduction of volume) is critical to facilitate the subsequent steps of purification. The capture step is commonly achieved by using chromatography with an ion-exchange resin. The drawback of using an ion-exchange resin is that the conductivity and/or pH of the loading has to be adjusted. When the conductivity is adjusted, in most instances reduced, it is performed by addition of water which increases the volume of the starting material, making it impractical for subsequent steps and overall cumbersome for production purposes. Furthermore, adjustment of pH often results in the formation of aggregates which could interfere with the performance of the purification steps.
After the capture step, an intermediate purification may follow, which removes most of the significant impurities including DNA, viruses and endotoxins. These impurities can also be remove/reduced in capture. The polishing step refers to a final purification step, wherein trace contaminants and impurities are removed and the yield is an active biological product. Contaminants removed during the polishing step are often conformers of the target molecule or suspected leakage products.
There is still a need in the art for improved purification methods which are fast and efficient and wherein Factor VIII activity is essentially retained.
The method of the present invention is advantageous in that it, in a single step, provides a volume reduction and a considerable increase in specific activity. Thus, the method, in a single step, combines a capture and a purification step.
The present invention also provides an efficient process for producing a highly concentrated and very pure solution of recombinant Factor VIII wherein the Factor VIII protein is stabilized against degradation. With the present invention, it is possible to combine a capture and purification step without risking severe destabilization of the FVIII molecules and, in a single step, obtain an initial purification of the crude sample, obtain a substantial volume reduction (thereby facilitating further purification steps) as well as obtain a substantial purification factor (increase in FVIII specific activity) and a resulting solution (“capture pool”) wherein the protein is stabilized against degradation.